Production of l-glutamic acid and alpha-ketoglutaric acids



United States Patent 3,120,472 PRODUCTIGN 0F L-GLUTAMIC ACID ANDa-KETOGLUTARIC ACIDS Cecil G. Dunn, Cambridge, Mass., George J. Fuld,Baltimore, MtL, Boguslaw W. Kusmierek, Wilmington, Del., Peter G. Lim,Bloomsburg, Pa., and Daniel I. C. Wang, Boston, Mass, assignors, bymesne assignments, to Hercules Powder Company, Wilmington, Del., acorporation of Delaware No Drawing. Filed Aug. 30, 1960, Ser. No. 52,755

7 Claims. (Cl. 19547) This invention relates to the biochemicalproduction of organic acids. In one aspect it relates to the productionof L-glutamic acid and a-ketoglutaric acid by cultivating microorganismshaving certain characteristics (as defined hereinafter) in a nutrientmedium under aerobic conditions, the medium including a carbohydratesource and a nitrogen source. More particularly, this invention relatesto the production of these acids by one of such microorganisms which wehave isolated as a new microorganism from farm soil. We have depositedthis microorganism with the Americal Type Culture Collection (ATCC), andthey have cataloged it under ATCC No. 13868. The characteristics givenin Table 1 hereinafter are based on extensive studies and tests,including, e.g., cultural, morphological, nutritional, physiological,and biochemical, which we have carried out on this microorganism. Weconsider that these characteristics fit the genus Corynebacterium betterthan they do any other known genus. However, these characteristics donot correspond suiiiciently to any known species of this genus. In viewof the foregoing we have designated this particular and preferredmicroorganism of our present invention as belonging to Corynebacteriumherculis spec. nov. It should be noted that the taxonomic area in whichthe microorganism ATCC No. 13868 and the other microorganisms of ourinvention are located has not been explored as extensively as some othertaxonomic areas; therefore, it is to be understood that our invention isnot limited to the use of microorganisms of any particular taxonomicdesignation except as set forth in the appended claims.

The present invention is applicable to the production of said organicacids with microorganisms characterized as aerobic, non-motile, non-acidfast, gram positive (by which term we mean to include gram variable),rod shaped bacteria which frequently are club shaped and often occur inangular arrangements. However, we

have obtained best results with Corynebacterium herculis spec. nov.,particularly in the production of L-glutamic acid. Also, at the presenttime L-glutamic acid is by far the more desirable acid commercially. Inview of the foregoing and for the sake of clarity and simplicity, the

present invention will be described hereinafter for the most part withrespect to the preparation of L-glutamic acid employingCorynebacterz'aum herculis spec. nov.

All known methods of making glutamic acid by chemical synthesis resultin a racemic or 50:50 mixture of L-glutamic acid and D-glutamic acidwhich is substanially useless as such. Only the L-glutamic acid byitself is of practical value, but the problems involved in separatingthe two acids make separation quite costly. The monosodium salt ofL-glutamic acid is widely used as flavor enhancer.

The following examples illustrate various embodiments of the presentinvention, but they are not intended to limit the invention beyond thescope of the claims of this application. In all examples, exceptExamples 9-12, 100-rnl. portions of an aqueous nutrient medium of aboutneutral pH in 500-ml. Erlenmeyer flasks were inoculated with 2-ml.portions of a liquid stock culture of the micro- 3,120,472 Patented Feb.4, 1964 "ice organism indicated and incubated at 28 C.-30 C. underareobic conditions on a rotary shaker with a speed of about 200oscillations per minute and a 1-inch diameter rotary motion. The stockcultures were considered to contain approximately 10 microorganisms perml.

Where the pH of the nutrient medium is not shown in the followingexamples, pH adjustment was not made because the nutrient medium wasalready approximately neutral in reaction.

In the examples and elsewhere herein the composition of the nutrientmedium is given as percent weight by volume except that theconcentration of flour wash water is given as percent volume by volume.

The flour wash water is, among others, a source of growth-promotingfactors, as discolsed in greater detail hereinafter. The flour washwater used is a by-product obtained in the well-known process ofseparating gluten and starch in flour by washing the flour with water.The flour wash water contains, among other things, soluble proteins,amino acids, vitamins, sugars, mineral salts, some material in thecolloidal state, and some small starch granules. The flour wash watersolids have the following approximate weight percent composition:

Protein 1 20 Sugar 38 Starch 20 Ash 10 Unaccounted for 12Nitrogen-containing materials calculated as proteins. Normally, thesolids content of the flour wash water is about 1%. For theseexperiments, except Examples 3 and 9, this was concentrated to 5% solidsby simple evaporation.

In the examples. the following methods of analyses were used todetermine the organic acids produced in accordance with this invention.An enzymatic method of analysis which involved a reaction catalyzed byL-glutamic acid decarboxylase and carried out in a Warburg apparatus wasused to determine the L-glutamic. acid produced. This is the method usedby the Glutamate Manufacturing Association and is often referred to asthe Warburg manometric method. This method is reported in Methods inEnzymology by Colowick and Kaplan, vol. 2, page 182, Academic Press,Inc., New York City (1955). The enzymatic method of analysis used todetermine the tt-ketoglutaric acid produced involved an enzyme-catalyzedtransamination reactionbetween ot-ketoglutaric acid and aspartic acid toyield glutamic acid and oxalacetic acid; oxalacetic acid was determinedby photometric analysis at 280 millimicrons.l. Biol. Chem. 234 1), page51 (1959). The paper chromatographic method used to determine theL-glutamic acid produced utilized a solvent system of isopropyl alcohol,phenol and water and a developer of 0.1% ninhydrin in acetone solution;the color was developed upon heating. The paper chromatographic methodused to determine the a-ketoglutaric acid utilized a solvent system ofbutyl alcohol, acetic acid and water and a developer of 0.2%o-phenylenediamine in ethyl alcohol solution containing 1% nitric acid;the color was developed on heating.

EXAMPLE 1 [C'orynebaeterium herculis spec. v. ATCC No. 13868]Composition of nutrient medium: Percent Glucose 4.68 KH PO 0.05 MgSO -7HO 0.02 Concentrated flour wash water (5% solids)--- 5.00 Urea 1.00

After 72 hours of fermentation the nutrient medium contained 18.6 mg. ofL-glu-tamic acid per of said medium.

3 EXAMPLE 2 [Coryncbacterium herculis spec. nov. ATCC No. 13868]Composition of nutrient medium Percent Glucose 0.936 KH PO 0.05 MgSO -7HO 0.02 Concentrated flour wash water solids) 5.00 Urea 1.00

After 72 hours of fermentation the nutrient medium contained 4.15 mg. ofL-glutamic acid per ml. of said medium.

EXAMPLE 3 [Goryncbactcrium herculis spec. nov. ATCC No. 13808]Composition of nutrient medium: Percent Glucose 9.36 KH PO 0.05

-Flour wash water (1% solids) 30.00 Total urea 2.20

After 168 hours of fermentation the nutrient medium contained 33.7 mg.of L-glutamic acid per ml. of said medium. In this example, instead ofall the urea being added at one time at the start of the fermentation,some was added then and the remainder was added subsequently in severalincrements during the fermentation.

EXAMPLE 4 [Coryncbaetcrium herculis spec. 110V. ATCC No. 13868]Composition of nutrient medium:

Glucose "percent" 4.68 KH PO do 0.238 K2HPO4 dO MgSO 7H O do 0.100 FeSO-7H O do 0.00011 MnCl -4H O do 0.00079 ZnSO -7H O do 0.00015 Urea do 0.8Yeast extract do 0.030 CaCO do 0.100 pH 7.0 After 72 hours offermentation the nutrient medium contained 12.1 mg. of L-glutamic acidper ml. of said medium.

EXAMPLE 5 [Coryncbuctcrizrm hcrcult's spec. nov. ATCC No. 13868]Composition of nutrient medium:

Glucose percent 4.68 Peptone do 0.5 Corn steep liquor do 0.2 KH PO do0.05 MgSO -7H O do 0.02 Urea do 0.8 pH 7.2

After 72 hours of fermentation the nutrient medium contained 7.8 mg. ofet-ketoglutaric acid per ml. of said medium.

EXAMPLE 6 [Coryncbacterium hercu'lis spec. nov. ATCC No. 13868]Composition of nutrient medium: Percent Glucose 4.68 KH PO 0.05 MgSO -7HO 0.02 Urea 1.0 Peptone 0.5 Corn steep liquor 0.2

After 48 hours of fermentation L-glutamic acid and a-ketoglutaric acidwere produced as determined by meth ods of analyses involving the use ofpaper chromatography, as disclosed hereinbefore. From the strength ofthe color developed it was apparent that the amount of etglutaric acidpresent was substantially greater than 4 the L-glutamic acid, but thatappreciable amounts of both acids were present.

After 48 hours of fermentation L-glutamic acid and a-ketoglutaric acidwere produced as determined by methods of analyses involving the use ofpaper chromatography, as disclosed hereinbefore. From the strength ofthe color developed it was apparent that the amount of L- glutamic acidpresent was substantially greater than the a-ketoglutaric acid, but thatappreciable amounts of both acids were present.

EXAMPLE 8 [Goryncbactcrium hcrculis spec. nov. ATCC No. 13868]Composition of nutrient medium: Percent Glucose 5.0 KH PO 0.05 MgSO -7HO 0.02 Urea 0.8 Distillers dry solubles 0.1

After 72 hours of fermentation the nutrient medium contained 7.1 mg. ofL-glutamic acid and 2.19 mg. of aketoglutaric acid per ml. of saidmedium.

EXAMPLE 9 [Goryncbacterium herculis spec. nov. ATCC No. 13808]Composition of nutrient medium: Percent Glucose 4.68 KH PO 0.10 K HPO0.20 MgSO -7H O 0.03 Total urea 1.0 Flour wash water (1% solids) 30.0

This example was carried out in substantially the same manner asExamples 1-8 above, except that it was on a much larger scale andutilized a stirred fermenter instead of shake flasks. The total urea wasadded in increments instead of all at the start of the fermentation.Thus 3 liters of the nutrient medium was inoculated with 300-ml. of theliquid stock culture of the microorganism. The aeration rate was 1.0volume air/volume nutrient medium/rninute.

After 72 hours of fermentation the nutrient medium contained 13.7 mg. ofL-glutamic acid per ml. of said medium.

EXAMPLES 10, 11 AND 12 [C'orynebacterium hcrculis spec. nov. ATCC No.13808] Composition of nutrient media:

Example N0 10 11 12 Percent Percent 0. 30 9. 30 0.03 0. 03 0.07 0.07 0.02 0. 02 0.03 0. 03 None None 1 1. 76 None Ammonium tartrate None I 2.

1 0.372% nitrogen equivalent.

In Examples 10, 11 and 12, 50-ml. of nutrient medium in 500-ml.Erlenmeyer flasks was used.

After 72 hours of fermentation substantial amounts of L-glutamic acidand a-ketoglutaric acid were produced.

In addition to the foregoing examples -we have practiced the presentinvention 'under substantially the same conditions as Examples 1-8above, except using biotin and thiamine as such as the growth-promotingfactors. Although We obtained substantial yields of L-glutanuc acid andn-ketoglutaric acid using biotin and thiamine as such, at present theother sources of growth-promoting factors disclosed herein are muchpreferred.

EXAMPLE 13 [Oorynebaeterium histidinolovorans ATCC No. 11442]Composition of nutrient medium:

Glucose percent 4.68 KI-I PO do 0.1 MgSO,- 7H O do 0.05 Urea do 0.8Yeast extract d 0.03 FeSO, do 0.000152 pH 7.0

After 72 hours of fermentation the nutrient medium contained L-glutamicacid as determined by the method of analysis involving the use of paperchromatography, as disclosed hereinbefore.

EXAMPLE 14 [C'orynebactem'mn fascians ATCC No. 12975] Composition ofnutrient medium: Percent Glucose 4.68 KH PO 0.05 MgSO -7H O 0.02 Urea1.0 Concentrated flour wash water solids) 5.0

the microorganism Corynebacterium herculis spec. nov.

ATCC No. 13868 in the production of the organic acids of this invention,microorganisms of the genus Corynebacterium may be used. Likewise, thenutrient medium may be varied considerably from the media shown in theexamples. We may use as a nitrogen source various materials, e.g. urea,inorganic ammonium salts including ammonium chloride and ammoniumsulfate and also organic ammonium salts including ammonium tartrate;ammonia itself is also applicable. Materials applicable as aicarbohydrate source include, e.g. glucose, hydrolyzed starch, sucrose,fructose.

We have found that the microorganisms of the present invention, ingeneral, require the addition of one or more factors for adequategrowth. For example, Table 2 given hereinafter illustrates how thegrowth requirement of Corynebacterium herculis spec. nov. ATCC No. 13868can be satisfied with biotin and thiamine as such. Therefore, in orderto cultivate StLlCh microorganisms for producing the acids of thepresent invention, such growth requirements must be satisfied. We havefound various sources (herein called growth-promoting factors sources)which satisfy these growth requirements. Materials applicable as asource of growth-promoting factors include, e.g. corn steep liquor,distillers dry solubles, peptone, yeast extract, flour wash water, crudeprotein hydrolyzates, bran solubles and vitamins such as thiamine andbiotin.

The amounts of the sources of carbohydrates, nitrogen, andgrowth-promoting factors are not critical. For optimum results theseamounts will depend on a number of factors, including the particularsource used. About 1%-15 carbohydrate source material gives goodresults, but somewhat better results are obtained using 5%-10% Inaddition, the amount of nitrogen source depends on the amount ofcarbohydrate source, so that for best results the amount of nitrogensource should be varied in the same direction as the variation in thecarbohydrate source. For example, when using 1%15% of carbohydratesource, say glucose, 0.5 %4.0% of nitrogen source material gives goodresults. The amount of growthpromoting factors source may vary widelyand is dependent for best results, among other things, on the particularsource employed.

Also, as is well known in this art, both the types and amounts of saltscan be varied quite widely. The present examples illustrate a largenumber of applicable salts. Perhaps the more important are the magnesiumand phos phate salts, and good results have been obtained with these inamounts of 0.01%-0.1% of MgSO and 0.05 0.8% of KH PO The pH range of thenutrient medium during fermentation should be about 5-9 and preferably7-8.

We prefer to operate at a fermentation temperature of about 30 C. Atsubstantially lower temperatures the rate of fermentation is slower. Inmost cases it would be undesirable to operate below about 25 C.Generally, there is no appreciable advantage in operating above about 37C. The microorganisms are apt to be seriously damaged, if not destroyed,at a temperature of about 50 C.

The duration of incubation or fermentation is not critical and may varyquite widely. It is dependent on a number of factors including theinooulum size and amounts of carbohydrate source material employed. Ingeneral, as the inoculum size increases, the fermentation timedecreases. Usually, as the amount of carbohydrate source materialincreases, the fermentation time increases. Fermentation time is alsodependent somewhat on temperature; the higher the tempertaure theshorter the fermentation time and vice versa. Within these conditions,the present invention is operable employing fermentation times of 12hours to 192 hours. However, usually about 48 hours to 72 hours willgive satisfactory results.

As disclosed hereinbefore, the monosodium salt of L- glutamic acid iswidely used as a flavor enhancer. oz- Ketoglutaric acid is used as aprecursor in the synthesis of L-glutamic acid.

Table 1 [Characteristics of Gorynebaetem'mn herculis spec nov. ATCC No.13868] Non-motile Gram positive Rod shaped with club shaped swellingsand angular arrangement of cells Many of the cells measure between about0.8-1.1 by

2.2 4.2 microns Agar colonies are punctiform, sometimes of about 2 mm.in diameter, smooth, entire, convex, opaque and of pale yellowish colorafter a few days 6. The agar stroke is characterized by moderate growth,filiform, glistening, light yellow, butyrous, no odor, medium unchanged7. Nutrient broth shows the following: moderate clouding, no surfacegrowth, no special odor, scanty vise-id sediment 8. Shows markeddiversity of form 9. Do not form spores such as characteristicallyformed by microorganisms of the genus Bacillus Contains granulesdemonstrable with methylene blue 11. Has growth-promoting factorsrequirements which can be satisfied with biotin and thiamine 12. Gelatinnot liquefied 13. Nitrite produced from nitrate 14. Aerobic 15. Catalasepositive 16. Ferments glucose, fructose, mannose, galactose, su-

crose, and arabinose with production of acid but no visible gas 17. Veryslight acid but no gases produced from dextrin,

and maltose 18. No acid and no gas produced from glycerol, lactose,spec. nov. ATCC No. 13868 which requires biotin and xylose, rhamnose,melibiose, mannitol, paraffin and thiamine in order to grow, saidcultivation being carried raffinose out until a substantial quantity ofL-glutamic acid is pro- 19. Nonthermoduric duced. 20. Does not hydrolzestarch 2. Process of preparing L-glutamic acid which com- 21. Grows wellat 30 C. and 37 C. prises cultivating under aerobic conditions in anutrient 22. Litmus milk: the reaction is "alkaline after days, mediumincluding a carbohydrate source, a nitrogen no peptonization, reduction,or curd formation 00- source, and a source of growth-promoting factorswhich curs supplies at least biotin and thiamine to the nutrient medi-23. Methyl red test positive 10 um Corynebacterium herculis spec. nov.ATCC No. 13868 24. VogesProsltaucr test: does not produce acctylmethylwhich requires biotin and thiamine in order to grow, said carbinolcultivation being carried out until a substantial quantity 25. Does notutilize citrate as a sole carbon source of L-glutarnic acid is produced.26. Produces hydrogen sulfide 3. Process of claim 2 wherein thecarbohydrate source 27. Non-acid fast is glucose. 28. Does not hydrolyzefat 4. Process of claim 2 wherein the carbohydrate source 29. Does notproduce indole is hydrolyzed starch.

T able 2 GROWTH STUDIES [Coryncbacterium Izcrculis spec. nov. ATCC No.13868] Run No 1 2 3 4 i 5 0 7 8 9 10 Compositionoi Nutrient Medium:

Glucose, percent 5.0 5.0 5.0 5. 0 5.0 5.0 5. 0 5.0 5. 0 5.0

KlIaPOi percenL. 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05

MgSO4.7HzO,pere 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02

Urea, percent 1.0 1. 0 1. 0 1. 0 1.0 1. 0 1. 0 1.0 1. 0 1.0

Biotin, millimierograms/mL. None 2.0 None 2.0 1.0 100 None None 1.0 100Thiamine,micrograms/ml None None 80 80 None None 10 1000 100 100 GrowthNo N o N 0 Yes N 0 N0 N 0 No Yes Yes In these growth studies thevitamins were added to the 5. Process of claim 2 wherein the nitrogensource is nutrient medium prior to inoculation. The nutrient rneurea.dium was inoculated 'with 0.1-ml. of an aqueous suspen 6. Process ofclaim 2 wherein the nitrogen source is sion of washed cells ofCorynebacterium herculis spec. ammonia. nov. ATCC No. 13868. In each rungrowth observa- 7. Process of claim 2 wherein the source of growthtionswere made several times during the last 54 hours of promoting factors isflour wash water. the 72-hour incubation period.

The L-glutamic acid and the a-ketoglutaric acid are References Cited inthe file of this Patent easily separated by conventional means, e.g. layion ex- 40 UNITED STATES PATENTS zharrliigiemtzchflrligzzf'or byfractional crystallization, from 3,002,889 Kinoshita et a1. Oct. 1961Since the microorganisms of this invention are aerobic, 3,087,863 L96 eta1 P 1963 they of course must be aerated. The type and extent of FOREIGNPATENTS aeration are interdependent with the other conditions of thepresent invention. From the present disclosure the 562,728 Canada Sept2, 1958 artisan will have no difiiculty insofar as aeration is con- OTHEREFERENCES cerned in practicing this invention.

As many apparent and widely diiferent embodiments of 23 Journal. ofBiochemistry (Japan) this invention may be made without departing fromthe 50 1 9 1 5 (1959) l 56 62 (1962) Chen.: Hakko Kogaku Zasshi 37,295-324 (1959) abspuit and scope thereof, it is to be understood thatthe tr t d Ch 1 Ab t t 54 13566 7 1960 i invention is not limited to thespecific embodiments i i l? sracs D (P Om fi gf i fg zijg g 35; L 55;Pat m 0 lzi osiiita et 131.1 lSulletin of the Agricultural Chemical i ae i i b p y e Society of Japan, vol. 22, N0. 3, pages 176485, May

1. Process of preparing organic acids selected from 1958 pmverslty ofTokyo 195-47' the group consisting of L-glutamic acid and a-ketoglutaAsa1 et al.: Journal of General Applied Microbiology acid whichcomprises cultivating 1mdm. aerobic condi (Tokyo), vol. 1, pp. 308-46(1955), abstracted in Chem1- tions in a nutrient medium including acarbohydrate cal fbflbstflfiwts, 52, 12311f (1958). source, a nitrogensource, and a source of growth-pro- Kmoshlta et Proceedings IntemationalSymposium moting factors which supplies at least biotin and thiamine tothe nutrient medium Corynebacterium herculis on Enzyme Chemistry, 1957,published by Maruzen (Tokyo), 1958, pp. 464-468.

1. PROCESS OF PREPARING ORGANIC ACIDS SELECTED FROM THE GROUP CONSISTINGOF L-GLUTAMIC ACID AND A-KETOG''UTARIC ACID WHICH COMPRISES CULTIVATINGUNDER AEROBIC CONDITIONS IN A NUTRIENT MEDIUM INCLUDING A CARBOHYDRATESOURCE, A NITROGEN SOURCE, AND A SOURCE OF GROWTH-PROMOTING FACTORSWHICH SUPPLIES AT LEAST BIOTIN AND THIAMINE TO THE NUTRIENT MEDIUMCORYNEBACTERIUM HERCULIS SPEC. NOV. ATCC NO. 13868 WHICH REQUIRES BIOTINAND THIAMINE IN ORDER TO GROWN, SAID CULTIVATION BEING CARRIED OUT UNTILA SUBSTANTIAL QUANTITY OF L-GLUTAMIC ACID IS PRODUCED.